Bang, Miseon
(Division of Animal Science, Chonnam National University)
,
Yong, Cheng-Chung
(Division of Animal Science, Chonnam National University)
,
Ko, Hyeok-Jin
(Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University)
,
Choi, In-Geol
(Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University)
,
Oh, Sejong
(Division of Animal Science, Chonnam National University)
Lactobacillus rhamnosus GG (LGG) is a probiotic commonly used in fermented dairy products. In this study, RNA-sequencing was performed to unravel the effects of acid stress on LGG. The transcriptomic data revealed that the exposure of LGG to acid at pH 4.5 (resembling the final pH of fermented dairy...
Lactobacillus rhamnosus GG (LGG) is a probiotic commonly used in fermented dairy products. In this study, RNA-sequencing was performed to unravel the effects of acid stress on LGG. The transcriptomic data revealed that the exposure of LGG to acid at pH 4.5 (resembling the final pH of fermented dairy products) for 1 h or 24 h provoked a stringent-type transcriptomic response wherein stress response- and glycolysis-related genes were upregulated, whereas genes involved in gluconeogenesis, amino acid metabolism, and nucleotide metabolism were suppressed. Notably, the pilus-specific adhesion genes, spaC, and spaF were significantly upregulated upon exposure to acid-stress. The transcriptomic results were further confirmed via quantitative polymerase chain reaction analysis. Moreover, acid-stressed LGG demonstrated an enhanced mucin-binding ability in vitro, with 1 log more LGG cells (p < 0.05) bound to a mucin layer in a 96-well culture plate as compared to the control. The enhanced intestinal binding ability of acid-stressed LGG was confirmed in an animal study, wherein significantly more viable LGG cells (${\geq}2log\;CFU/g$) were observed in the ileum, caecum, and colon of acid-stressed LGG-treated mice as compared with a non-acid-stressed LGG-treated control group. To our knowledge, this is the first report showing that acid stress enhanced the intestine-binding ability of LGG through the induction of pili-related genes.
Lactobacillus rhamnosus GG (LGG) is a probiotic commonly used in fermented dairy products. In this study, RNA-sequencing was performed to unravel the effects of acid stress on LGG. The transcriptomic data revealed that the exposure of LGG to acid at pH 4.5 (resembling the final pH of fermented dairy products) for 1 h or 24 h provoked a stringent-type transcriptomic response wherein stress response- and glycolysis-related genes were upregulated, whereas genes involved in gluconeogenesis, amino acid metabolism, and nucleotide metabolism were suppressed. Notably, the pilus-specific adhesion genes, spaC, and spaF were significantly upregulated upon exposure to acid-stress. The transcriptomic results were further confirmed via quantitative polymerase chain reaction analysis. Moreover, acid-stressed LGG demonstrated an enhanced mucin-binding ability in vitro, with 1 log more LGG cells (p < 0.05) bound to a mucin layer in a 96-well culture plate as compared to the control. The enhanced intestinal binding ability of acid-stressed LGG was confirmed in an animal study, wherein significantly more viable LGG cells (${\geq}2log\;CFU/g$) were observed in the ileum, caecum, and colon of acid-stressed LGG-treated mice as compared with a non-acid-stressed LGG-treated control group. To our knowledge, this is the first report showing that acid stress enhanced the intestine-binding ability of LGG through the induction of pili-related genes.
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제안 방법
85% NaCl to remove the remaining FITC and then orally administered to male C57BL/6J mice. After 12 h, the mice were sacrificed, and their intestines were collected to detect the presence of adherent labeled cells using a confocal laser scanning microscope (FV500, Olympus, Japan). Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Chonnam National University (project number: CNUIACUC-YB-2012-40).
Quantitative PCR (qPCR) was performed using KAPA SYBR FAST qPCR kit (KapaBiosystems, USA) in a thermal cycler (Bio-Rad Laboratories, USA) with the conditions of initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 sec, annealing at 54°C for 15 sec and extension at 72°C for 15 sec.
The findings presented in this study provide a detailed understanding of the adaptation of LGG towards acid stress conditions, including the enhancement of its adhesion ability (Fig. 6). Our findings lead us to a novel discovery regarding the enhanced adhesion properties of LGG upon acid stress treatment, especially the expression of the spaFED pili gene cluster, which was previously undetectable in LGG cultured outside the mammalian gastrointestinal tract.
대상 데이터
RNA sequencing was performed with an Illumina MiSeq platform. All data sets have been deposited in the Gene Expression Omnibus database under accession number GSE107337. Data analysis was performed using EdgeR, Bioconductor components in R packages with LGG as the reference genome.
Lactobacillus rhamnosus strain GG ATCC 53103 was obtained from Valio, Helsinki, Finland, and maintained in de-Man, Rogosa and Sharpe (MRS) medium to mid-log phase (optical density at 600 nm [OD600nm] of 0.6, equivalent to approximately 3 × 108 CFU/ml).
cDNA was synthesized from 1 µg of the extracted RNA with an RT Premix Oligo (dt) Kit (Intron, Korea). The primers used in this study were designed using Primer3plus software (http://www. bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), based on the genome sequence of LGG (Table 1). Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as an internal control gene.
데이터처리
Duncan’s multiple range test was used to determine the significance of differences between means at a significance threshold of p < 0.05.
성능/효과
Based on our results, exposure to acid stress conditions similar to those in fermented dairy products enhanced the adhesion properties of LGG, to confer a competitive advantage in the mucosal environment for which to outcompete pathogens with advanced probiotic properties.
Owing to the inability of in vitro assays to resemble the complex environmental conditions of cells within a living organism, an animal study was conducted to evaluate the effect of acid stress on the adherence and retention of LGG during transit through the gastrointestinal tract. The results of the in vivo study confirmed the enhanced adhesion of LGG in the gastrointestinal tract under acid stress conditions, indicating that acid stress could enhance the adhesion of LGG and thereby prolong its retention in the gastrointestinal tract, enabling it to exert its health-promoting properties.
To assess the reliability of the transcriptomic and gene expression analyses, the mucin-binding ability of LGG upon acid stress was investigated. The results showed a positive correlation between the transcriptomic and gene expression data and the mucin-binding ability of LGG. The number of LGG cells attached to the mucin layer was 1 log CFU/ml higher (p < 0.
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